Methods and cell line useful for production of recombinant adeno-associated viruses

ABSTRACT

Methods for efficient production of recombinant AAV employ a host cell which comprising AAV rep and cap genes stably integrated within the cell&#39;s chromosomes, wherein the AAV rep and cap genes are each operatively linked to regulatory sequences capable of directing the expression of the rep and cap gene products upon infection of the cell with a helper virus, a helper gene, and a helper gene product. A method for producing recombinant adeno-associated virus (rAAV) involves infecting such a host cell with a helper virus, gene or gene product and infecting the infected host cell with a recombinant hybrid virus or plasmid vector containing adenovirus cis-elements necessary for replication and virion encapsidation, AAV sequences comprising the 5′ and 3′ ITRs of an AAV, and a selected gene operatively linked to regulatory sequences directing its expression, which is flanked by the above-mentioned AAV sequences.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This is a continuation of U.S. patent application Ser. No.09/528,017, filed Mar. 17, 2000, which is a continuation ofInternational Patent Application No. PCT/US98/19463, filed on Sep. 18,1998, which claims the benefit of the priority of U.S. patentapplication Ser. No. 60/059,340, filed on Sep. 19, 1997.

[0002] This invention was made with financial assistance from theNational Institutes of Health Grant Nos. NIAMS P01AR/MS43648, P30DK47757-05, P01 HD32649-04, P01 AR/NS43648-03, and P01 CA66726-03. TheUnited States government has certain rights in this invention.

BACKGROUND OF THE INVENTION

[0003] Adeno-associated virus (AAV) is a replication-deficient humanparvovirus, the genome of which is about 4.6 kb in length, including 145nucleotide inverted terminal repeats (ITRs). Two open reading framesencode a series of rep and cap polypeptides. Rep polypeptides (rep78,rep68, rep62 and rep40) are involved in replication, rescue andintegration of the AAV genome. The cap proteins (VP1, VP2 and VP3) formthe virion capsid. Flanking the rep and cap open reading frames at the5′ and 3′ ends are 145 bp inverted terminal repeats (ITRs), the first125 bp of which are capable of forming Y- or T-shaped duplex structures.Of importance for the development of AAV vectors, the entire rep and capdomains can be excised and replaced with a therapeutic or reportertransgene [B. J. Carter, in “Handbook of Parvoviruses”, ed., P. Tijsser,CRC Press, pp.155-168 (1990)]. It has been shown that the ITRs representthe minimal sequence required for rescue, packaging, and integration ofthe AAV genome.

[0004] The wild type, nonpathogenic human virus is capable of infectinga wide variety of cells and establishing a latent infection of the cellvia a provirus that integrates at high frequency into a specific regionof chromosome 19 [Kotin, R. M. et al, Proc. Natl. Acad. Sci. USA 87,2211-2215 (1990); Samulski, R. J. et al. EMBO J. 10, 3941-3950 (1991)].Production of infectious virus and replication of the virus does notoccur unless the cell is coinfected with a helper virus, such asadenovirus or herpesvirus. Upon infection with a helper virus, the genesof latent AAV (i.e., rep and cap) are activated, resulting in rescue ofthe AAV provirus, replication of the AAV genome, and formation of AAVvirions, as well as generation of additional helper virus. The infectingparental ssDNA is expanded to duplex replicating form (RF) DNAs in a repdependent manner. The rescued AAV genomes are packaged into preformedprotein capsids (icosahedral symmetry approximately 20 nm in diameter)and released as infectious virions that have packaged either+ or −ss DNAgenomes following cell lysis.

[0005] AAV possesses unique features that make it attractive as a vectorfor delivering foreign DNA to cells. Progress towards establishing AAVas a transducing vector for gene therapy has been slow. Evaluation ofrecombinant AAV (rAAV) produced recombinantly and exclusive of wildtypeAAV reformed by recombinant methods in preclinical models of genetherapy has been limited for a variety of reasons, primarily becausemethods of production are inefficient and often generate substantialquantities of replication competent AAV. Replication defective forms ofAAV are created by transfecting vector DNA (transgene flanked by AAVITRs) together with a rep/cap expressing plasmid concurrent withadenovirus infection [Samulski, R. et al, J. Virol, 61(10):3096-3101(1987)] or transfection with an adenovirus helper plasmid [Ferrari, F.K. et al, Nature Med., 3, 1295-1297 (1997)]. The standard method, basedon transient transfection, is not easily scaled-up, making it difficultto obtain virus [Fisher, K. J. et al. J. Virol. 70, 520-532 (1996)].Furthermore, preparations are invariably contaminated with replicationcompetent AAV (rcAAV) formed by, for example, nonhomologousrecombination, during the process of transfection. Another method forproducing rAAV has been described based on the simultaneous transienttransfection of cis and trans plasmids together with an adenovirushelper plasmid [Ferrari et al, Nature Med. 3: 1295-1297 (1997)]. Thisapproach has the advantage of minimizing contaminating adenovirus butsuffers from the creation of rcAAV and difficulties in scaling-up. Aspecific obstacle to the use of AAV for delivery of DNA, especially fortherapeutic applications, has been lack of highly efficient schemes forencapsidation of recombinant genomes and production of infectiousvirions [R. Kotin, Hum. Gene Ther., 5:793-801 (1994)].

[0006] Problems exist in attempts to improve AAV production includingthe use of cell lines stably expressing vector and/or rep/cap. Creationof a cell line stably expressing rep and cap has been difficult becauseof cellular toxicity of rep gene products. Several cell lines previouslydescribed do not express sufficient quantities of rep or cap to sustainhigh titer production of vector [Tamayose, K. et al, Hum. Gene Thera. 7,507-513 (1996); Yang, Q., et al, J. Virol. 68, 4847-4856 (1994); see,also, Clark, K. R. et al, Hum. Gene Thera. 6, 1329-1341 (1995) and U.S.Pat. No. 5,658,785]. Other strategies have been described that providemore efficient ways to transiently express and regulate rep and cap[Mamounas, M., et al, Gene Thera. 2, 429-432 (1995); and Flotte, T.R. etal. Gene Thera. 2, 29-37 (1995)].

[0007] Disadvantages of current methods for production of rAAV thatemploy transfection of rAAV genome into host cells followed byco-infection with wild-type AAV and adenovirus, include the productionof unacceptably high levels of wild-type AAV, little recombinant geneexpression and inefficient integration. Another recognized means formanufacturing transducing AAV virions entails co-transfection with twodifferent, yet complementing plasmids. One of these plasmids contains atherapeutic or reporter transgene flanked (sandwiched) between the twocis acting AAV ITRs. The AAV components that are needed for rescue andsubsequent packaging of progeny recombinant genomes are provided intrans by a second plasmid encoding the viral open reading frames for repand cap proteins. However, both rep and cap are toxic to the host cells.This toxicity has been the major source of difficulty in providing thesegenes in trans for the construction of a useful rAAV gene therapyvector.

[0008] There remains a need in the art for additional methods permittingthe efficient production of AAV and recombinant AAV viruses for use asvectors for somatic gene therapy at high titers not previously achieved.

SUMMARY OF THE INVENTION

[0009] The present invention provides a novel method and a novel cellline which permits efficient and high level production of recombinantadeno-associated virus (rAAV) as described in detail below, and producesrAAV essentially free of replication competent AAV (rcAAV).

[0010] In one aspect, the invention provides a cell comprising an AAVrep gene and an AAV cap gene stably integrated within the cell'schromosomes, wherein the AAV rep and cap genes are operatively linked toregulatory sequences capable of directing the expression of the rep andcap genes, and wherein the cell expresses gene products of the rep andcap genes upon introduction to the cell of a helper. The helper is ahelper virus, a helper gene, or a helper gene product. The cell lines ofthis invention are characterized by integration of multiple copies ofpromoter-rep-cap gene cassettes in a concatamer form into the hostchromosome. The cell lines of this invention are also characterized byproviding high level expression of rAAV (e.g., greater than 1×10³ rAAVparticles per cell) upon the introduction of the helper to the cell linein comparison to the yields of rAAV from other stably rep/captransfected cells. One embodiment of this cell is a host cell derivedfrom HeLa cells, B-50 [ATCC Accession No. CRL-12401] which stablyexpresses AAV rep and cap genes under the control of the endogenous AAVp5 promoter.

[0011] In another aspect, the invention provides a method for producinga helper-infected host cell. The method includes the step of introducingto a host cell a helper, the host cell comprising an AAV rep gene and anAAV cap gene stably integrated within the host cell's chromosomes,wherein the AAV rep and cap genes are each under the control ofregulatory sequences capable of directing the expression of the rep andcap genes, and wherein the host cell expresses products of the rep andcap genes upon introduction to the host cell of the helper. The helpercomprises a helper virus, a helper gene, or a helper gene product.

[0012] In another aspect, the invention provides a method for producingrecombinant AAV, which includes the step of introducing to the host cellof this invention which contains the helper as described immediatelyabove, a recombinant hybrid virus. The recombinant hybrid viruscomprises a selected transgene operatively linked to regulatorysequences controlling the transgene's expression. The transgene withlinked regulatory sequences is flanked by AAV sequences comprising the5′ and 3′ ITRs of an AAV. The 5′ ITR flanks one side of the transgene,and the 3′ ITR flanks the other side. The transgene with linkedregulatory sequences and with flanking AAV sequences is further flankedby at least one adenovirus cis-element. The cis elements useful in thismethod are the cis elements required for replication of adenovirusvirions and the cis elements required for encapsidation of adenovirusvirions. The method optionally includes the additional step of isolatingfrom the hybrid-virus-infected helper-infected host cell a recombinantAAV, the recombinant AAV comprising the transgene. This method permitsrecombinant AAV to be produced by the cell.

[0013] In one embodiment of the aspects of this invention, a method forproducing recombinant AAV is referred to as the B50/hybrid method. Thismethod includes the steps of infecting one of the host cells describedherein with a helper (e.g., virus, gene or gene product) to induce AAVrep and cap expression and provide necessary helper functions, andtransfecting the infected host cell with a recombinant adenovirus-AAVhybrid virus, which carries a selected transgene. In one desirableembodiment, production of AAV occurs in a two step process: A host cellof this invention which stably expresses rep and cap (e.g., B-50) isinfected with helper, e.g., an adenovirus preferably defective in E2b,followed by infection with another virus, e.g., a hybrid virus, oneexample of which is an AdAAV hybrid in which an AAV vector containing atransgene under the control of regulatory sequences, is cloned in the E1region of a replication defective adenovirus. This results in a 100-foldamplification and rescue of the AAV genome, leading to high yield ofrecombinant AAV that is free of rcAAV.

[0014] Other aspects and advantages of the present invention aredescribed further in the following detailed description of the preferredembodiments thereof

BRIEF DESCRIPTION OF THE DRAWINGS

[0015]FIG. 1 is a graph illustrating the productivity of a rAAV carryinga LacZ transgene in B-50 host cells which were infected with wildtypeadenovirus type 5 (Ad5wt) and transfected with a hybrid virus accordingto this invention. The symbols represent hours post Ad5wt infection: 0hours (□); 8 hours (open diamond); 12 hours (◯); 16 hours (Δ); 20 hours(cross-hatched square); 24 hours (cross-hatched diamond).

[0016]FIG. 2A is a bar graph demonstrating amplification of AAV genomein the hybrid shuttle virus Ad.AVCMVLacZ (also Ad-AAVlacZ) and theimpact of infection sequence on vector production in B50 cells, asreported in Example 2. The production of rAAV under different conditionsis represented infectious units referred to as lacZ forming units (LFU)per cell vs. time interval between infection by the helper virus andinfection with the hybrid virus.

[0017]FIG. 2B is a graph showing amplifications of hybrid virus and rAAVin B50 cells which were infected with Ad5wt 24 hours prior to Ad-AAVlacZat a multiplicity of infection (MOI) of 10. See the detailed protocol ofExample 2. The dotted line represents growth kinetics of the hybridwhich is defined as the transduction measured after heat inactivation.The solid line indicates excision and amplification of rAAV genomes andproduction of vector as measured by transduction after heatinactivation.

[0018]FIG. 3A is a graph showing the analysis of mice followinginfection of a rAAV carrying an erythropoietin gene (AAV-Epo) intoskeletal muscle as described in Example 5. Mice received AAV-Epo derivedfrom conventional 293/cotransfection (squares) or AAV-Epo derived fromuse of one embodiment of a cell line of this invention and a method ofthis invention (triangles). Epo levels are measured as 1 μ/L.

[0019]FIG. 3B is a graph measuring the hematocrits as % blood volume foranimals treated as described in Example 5.

DETAILED DESCRIPTION OF THE INVENTION

[0020] The invention provides methods and compositions for theproduction of recombinant adeno-associated virus (rAAV) in yields ofgreater than about 1×10³ viral particles per cell to much higher viralparticle yields, as described below. The method of this invention can bedesirably employed to produce rAAV carrying transgenes, which correct adefect in a cell to modulate or alleviate the symptoms associated withthe defect. These methods are particularly useful in transferring thetransgene to a host cell or tissue. These rAAV are useful as researchreagents, as tools for the recombinant production of a transgene productin vitro, and as tools for the production of gene therapy reagents.

[0021] I. Definitions

[0022] Recombinant AAV (rAAV) as used herein, is defined as a structurecontaining an AAV capsid, necessary AAV cis elements, and a heterologousgene (i.e., a transgene) of interest which is under the control ofregulatory sequences (e.g., a promoter and/or regulator sequence) whichdirect the expression of the product of the gene under suitableconditions. Suitably the AAV cis elements include the AAV 5′ and 3′inverted terminal repeats sequences (ITRs). The rAAV is devoid of therep gene and other AAV structural genes. The rAAV is not itself capableof replication. The term rAAV encompasses functional modifications tothe AAV cis elements

[0023] Replication competent AAV (rcAAV) is defined herein as any AAVwhich can replicate in the presence of a helper which can transactivatethe AAV rep and cap genes, e.g., an adenovirus helper. rcAAV mustcontain rep and cap. Thus, examples of rcAAV include wildtype (wt) AAV,modified wtAAV resulting from, e.g., unwanted homologous recombination,or a rearranged AAV containing a portion of a transgene resulting from,e.g., unwanted homologous recombination, so as to accommodate the repand cap genes.

[0024] The terms “genome copies” and “particle number” areinterchangeable, and are measurements of productivity (or yield) of apackaging cell line of this invention. These terms refer to the averagenumber of rAAV virus particles that can be produced per host cell orproduction/packaging cell line, and is correlated to infectious units.The genome copy or particle number is a measurement of AAV DNA only,without regard to whether the AAV particle is infectious. The yield(i.e., the genome copy) is not influenced by concentration orpurification. For evaluation of a cell line, the higher the genome copyor particle number, the more productive the cell. Cells of the presentinvention are characterized by genome copies greater than 1×10³. Othercells of this invention are characterized by genome copies greater than5×10³. Still other cells of the present invention are characterized bygenome copies greater than 1×10⁴. Other cells of this invention arecharacterized by genome copies greater than 5×10⁴. Cells of the presentinvention are characterized by genome copies greater than 1×10⁵. Othercells of this invention are characterized by genome copies greater than5×10⁵. Still other cells of the present invention are characterized bygenome copies greater than 1×10⁶. Wherever in the following description,a cell of this invention is characterized by the phrase “high yield” or“efficient production”, such phrases are defined numerically by thegenome copy numbers above.

[0025] Infectious Unit (IU) or Infection Forming Unit (IFU), as usedspecifically herein, provides a measurement of the ability of an rAAVparticle to infect a cell. One IU is equivalent to one LacZ forming unit(LFU), which is a term applied only to the rAAV harboring the transgenebeta-galactosidase. IU can be measured either with or without thepurification process which separates adenovirus or other helper virusfrom the rAAV. IU can be affected by purification or concentration. Thesmaller the IU, the more infectious is the AAV particle. As used in thisspecification, one IU or one LFU is equivalent to less than about 1×10⁶viral particles. In other embodiments of this invention, one IU isequivalent to less than 1×10⁵ viral particles. More desirably, one IU isequivalent to less than 1×10⁴ particle numbers. Preferably one IU isequivalent to less than 1×10³ particle numbers.

[0026] The term “genome titer” generally refers to the genome copy orparticle number per milliliter.

[0027] The term “transducing unit” or “transduction unit” is definedherein as the number of cells transduced with infectious rAAV. This termis also referred to as the potency of rAAV. Each infected cell maycontain greater than one infectious AAV particle.

[0028] II. Host Cells of the Invention

[0029] A cell or host cell of the present invention is a cell,preferably a mammalian cell, that comprises an AAV rep gene and an AAVcap gene stably integrated within the cell's chromosomes. The host cellitself is preferably a mammalian cell, of which many suitable types arewell-known in the art. Suitable parental cell lines which can be used toprepare host cells and host cell lines of this invention include,without limitation, HeLa [ATCC CCL2], A549 [ATCC Accession No. CCL 185],KB [CCL 17], Detroit [e.g., Detroit 510, CCL 72] and WI-38 [CCL 75]cells. These cell lines are all available from the American Type CultureCollection, 10801 University Boulevard, Manassas, Va. 20110-2209 USA.

[0030] The AAV rep and cap genes in the host cell may be obtained fromamong the many known serotypes of AAV. Both genes may be derived fromthe same AAV serotype or from different AAV serotypes. The rep and capgenes in the host cell of the invention are operatively linked toregulatory sequences capable of directing the expression of the rep andcap genes. The rep gene may be operatively linked to a differentregulatory sequence than that directing the expression of the cap gene.Alternatively, the rep and cap genes may be operatively linked to thesame regulatory sequences in the host cells. Such regulatory sequencesare conventional and include promoters and other sequences which controltranslation and expression of the gene products. These regulatorysequences may be exogenous to the host cell.

[0031] The regulatory sequences may include constitutive promoters orregulated (inducible) promoters, which will enable controlled expressionof the rep/cap. For example, one promoter is the liver specific albuminpromoter. Another desirable promoter is a β-actin promoter, which isdesirably used in combination with a cytomegalovirus (CMV) enhancer.Still other non-AAV promoters include, without limitation, the Roussarcoma virus LTR promoter/enhancer, the cytomegalovirus immediate earlypromoter/enhancer [see, e.g., Boshart et al, Cell, 41:521-530 (1985)],and the inducible mouse metallothienien promoter. Still otherpromoter/enhancer sequences may be selected by one of skill in the art.

[0032] However, in a preferred embodiment, the regulatory sequencesinclude AAV regulatory sequences, such as the AAV p5 promoter. The AAVregulatory sequences, e.g., the p5 promoter, may be derived from adifferent AAV serotype than that which provided the rep and cap genes.Alternatively, the same AAV serotype may provide all of the AAVcomponents of the host cell.

[0033] The cells of the present invention are also characterized by thepresence of the rep and cap genes in multiple copies stably integratedwithin the cell's chromosomes. These multiple copies may be present inthe chromosomes in concatameric form, i.e., head-to-tail or head-to headorder. Thus, cells of the present invention are characterized by thepresence of the rep and cap genes in at least two copies stablyintegrated within the cell's chromosomes. Other cells of the presentinvention are characterized by the presence of the rep and cap genes inat least three copies stably integrated within the cell's chromosomes.Still other cells of the present invention are characterized by thepresence of the rep and cap genes in at least four copies stablyintegrated within the cell's chromosomes. An embodiment of a cell ofthis invention described below contains five copies of rep/cap stablyintegrated with the cell's chromosomes. These multiple copies may alsocontain repeated copies of the promoter/regulatory sequences whichcontrol expression of the rep/cap.

[0034] The cells of this invention express the gene products of the repand cap genes upon introduction to the cell of a helper. Appropriatehelpers are identified in the description of the methods below.

[0035] When appropriate helpers and hybrid viruses are introduced to thecells of the present invention, according to the methods described indetail in part III below, the cells of the present invention arecharacterized by efficient production of high yields of rAAV in contrastto the productivities of packaging cells of the prior art. When used toproduce rAAV in the below-defined methods, the productivity of cells ofthis invention may be defined as producing yields of rAAV greater than1×10³ genome copies per cell. Cells of this invention are alsocharacterized by producing at least 3×10³ genome copies per cell. Othercells of this invention are characterized by yielding at least 1×10 ⁴genome copies per cell. Still other cells of this invention arecharacterized by producing greater than 5×10⁴ genome copies per cell.Other cells of this invention are characterized by producing yields ofgreater than 1×10⁵ genome copies per cell. Cells of this invention arecharacterized by producing greater than 5×10⁵ genome copies per cell.The cells of this invention are characterized by genome copies ofgreater than 1×10⁶ genome copies per cell.

[0036] One illustrative embodiment of a host cell of this invention isB-50. This B-50 cell line was deposited with the American Type CultureCollection, 10801 University Boulevard, Manassas, Va., United States ofAmerica 20110-2209, USA 20852 on Sep. 18, 1997 under Accession numberCRL12401 pursuant to the requirements of The Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurposes of Patent Procedure. B-50 is a HeLa cell line, which, upon theintroduction of a helper, stably expresses AAV type 2 rep and cap genesunder the control of the homologous p5 promoter. B-50 is characterizedby integration of multiple copies (at least 5 copies) of P5-rep-cap genecassettes in a concatamer form into the host chromosome. B-50demonstrates stable, efficient and high yield expression of rep and capupon adenovirus infection. B-50 is an improvement over other stablerep/cap expressing cell lines, such as those described in U.S. Pat. No.5,658,785, because of its unusually and unexpectedly high level ofexpression of the rep and cap gene products. The expression level andprotein profile of rep/cap in B-50 cells infected with adenovirus helperare between 5-fold to 10-fold better than those attributes of HEK293cells transfected with pAd/AAV, a plasmid construct containing theentire AAV coding sequences including p5 promoter [Samulski, R. J. etal, J. Virol., 63:3822-3828 (1989)] and being widely used for rAAVproduction by classical transfection methods.

[0037] The host cells of this invention, including e.g., B-50 cells, canbe used not only for large scale production of rAAV through the methodsof this invention described in detail below, but also for generatingrAAV producer cell lines with a gene of interest. In addition, the cellsof this invention have many other applications such as titering rAAVpreparation in assays, e.g., an Infectious Center Assay [Snyder, R. O.et al, “Production of recombinant adeno-associated viral vectors. ”, inCurrent Protocols in Human Genetics, Vol. 1, (eds. Dracopoli, N. et al.)pp 1-24 (John Wiley & Sons, NY 1996)] and as research tools for studyingthe biological functions of rep/cap proteins, promoter influence, cellcycle regulation, and the like.

[0038] III. Method of the Invention

[0039] The present invention also provides methods for producing ahelper-infected host cell and methods for producing the aforementionedhigh yields of rAAV by using the infected host cells of the invention.These methods of the present invention provide a response to the needfor gene delivery vectors which may be efficiently produced in highyields and which are substantially purified. The methods of the presentinvention do not require transient transfection, but instead utilizeintroduction to the cell line of the present invention a helper.

[0040] According to these methods, a helper is introduced to a host cellas described above, i.e., a mammalian cell comprising an AAV rep geneand an AAV cap gene stably integrated within the host cell'schromosomes, wherein the rep and cap genes are each under the control ofregulatory sequences capable of directing the expression of the rep andcap genes. In the presence of the helper, the host cell expresses thegene products of the rep and cap genes.

[0041] The helper may be in the form of a helper virus, or a plasmid; orit may be delivered as a helper gene, or a helper gene product or assome other construct. The helper is capable of providing helperfunctions (i.e., capable of activating the expression of the rep and capgenes in the host cell) in any of its forms. Use of the term “helpervirus” in this application means any of these elements capable ofproviding helper functions. Thus, in one example, the helper includesthe adenovirus E1 gene or gene product. The E1 gene may be delivered tothe cell by infection with an E1-containing adenovirus, e.g., awild-type or otherwise modified E1-containing adenovirus, or arecombinant adenovirus or a plasmid containing E1. The gene product maybe delivered directly to the cell. Alternatively, the transactivatinghelper may be another virus (not an adenovirus) which contains theadenovirus E1 gene and functions to deliver the AdE1 gene to the cellline. Such other virus may be selected from among a number of knownviruses commonly employed to vector exogenous genes to cells.

[0042] Still another alternative transactivating helper is a herpesvirus, which also has the ability to activate rep/cap expression from anAAV P5 promoter, among others. The helper may be a wildtype herpesvirus, a recombinant herpes virus, a herpes virus gene, or a herpesvirus gene product. The gene product may be delivered directly to thecell. Among suitable herpes viruses are Herpes simplex I and HerpesSimplex II. Also useful as transactivating helper viruses are Vacciniaand Cytomegalovirus. These viruses may be modified or replicationdefective.

[0043] Alternatively, the helper virus may be a temperature sensitivemutant virus which will be self-replication defective at non-permissivetemperatures, and therefore, will only provide necessary helperfunctions to rAAV production.

[0044] As reported in the prior art, those adenovirus genes necessaryfor maximal AAV production levels appear to include E1, E2a, E4 and VAI[Kotin, R. M. Hum. Gene Thera. 5, 793-801 (1994)]. Such other helpergenes which are necessary for rAAV production may be introduced to thecell in the same manner as E1, described above, i.e., in the same helperconstruct or virus. Alternatively, helper genes other than thetransactivating helper, e.g., E1, may be delivered at some time afterthe transactivating helper by introducing an additional helper to thecell. For example, the helper genes other than E1 may be delivered tothe host cell via a replication-defective adenovirus, among otherhelpers.

[0045] Once the helper, or at least the transactivating helper isintroduced to the cell, it may employed in a method for producingrecombinant AAV. The method of this invention is based on infecting thehelper-containing, rep/cap expressing cell line of the invention withrecombinant hybrid virus, e.g., an Ad-AAV hybrid virus, whichsubstantially increases the yield of rAAV and simplifies scale-up,allowing the study of multiple subjects from a single preparation ofrAAV. The recombinant hybrid virus comprises a selected transgeneoperatively linked to regulatory sequences controlling the transgene'sexpression, the transgene with linked regulatory sequences being flankedby AAV sequences comprising the 5′ and 3′ ITRs of an AAV, wherein the 5′ITR flanks one side of the transgene, and the 3′ ITR flanks the otherside. The transgene with linked regulatory sequences and with flankingAAV sequences is flanked by at least one adenovirus cis-element. Thecis-element is selected from among cis elements required for replicationof adenovirus virions and cis elements required for encapsidation ofadenovirus virions.

[0046] One embodiment of a hybrid AdAAV virus which can be used in themethods of this invention is described in detail in International PatentPublication No. WO96/13598, published on May 9, 1996, and incorporatedby reference herein. Essentially, the AdAAV hybrid virus or a hybridvirus vector (plasmid) as described above provides in cis a minigene,which comprises a selected heterologous transgene under the control ofregulatory sequences directing expression thereof in the host cell.

[0047] The introduction of the hybrid virus or vector into the host cellis accomplished using known techniques. The use of the hybrid virus,which substantially amplifies the AAV DNA prior to rescue andreplication, is believed to assist in generating high yields of rAAVproduced by the methods of this invention.

[0048] The introduction of a helper which can transactivate (e.g.,activate the promoter controlling expression of rep/cap in the cell) therep/cap expression in the stable rep/cap expressing cell line of thisinvention and provide helper functions for AAV replication, followed bythe introduction of the Ad-AAV hybrid virus provides the componentsnecessary for rAAV production.

[0049] Recombinant AAV comprising the transgene are produced by the celland are isolated therefrom in high yields as identified above (e.g., therecombinant AAV is produced at levels exceeding 1×10³ genome copies percell). Also, e.g., the rAAV may also be produced at a level exceeding1×10⁶ genome copies per cell. These recombinant AAV are essentiallyhomogenous, that is, the rAAV produced by the method of this inventionare essentially free of replication-competent AAV. See the embodimentdiscussed in Example 4 below.

[0050] In the methods described herein, the temporal relationshipbetween rep/cap induction and AAV rescue and replication as well asinfectious dose, i.e., the multiplicity of infection (MOI) of the helperand AdAAV hybrid viruses, can be readily adjusted to optimize rAAVproduction, depending on the cell line, helper(s) and AdAAV hybrid used.Such adjustments are accomplished by performing experiments varying thetimes of helper introduction(s), hybrid virus infection and MOIs ofhelper(s) and hybrid. See, e.g., the experiments described above andreported in FIGS. 2A and 2B and in the examples below. For example, inthe embodiments described in FIG. 2A, for example, the time betweeninfection of the wild type and hybrid viruses was varied, demonstratinga substantial increase in rAAV production with maximal yields obtainedwhen a cell of this invention was infected with hybrid virus between 12to 36 hours after the helper was introduced to the cells. In thatparticular embodiment of FIG. 2A, the introduction of hybrid virus 24hours after the introduction of helper was optimal. Balancing dosage andtemporal aspects of these methods are routine protocols and may beperformed to determine the best conditions for the parameters of themethod. Such protocols as described in the Examples below are skillswell within the art.

[0051] In one particular embodiment of a method of this inventionreferred to as the B-50/hybrid method, the B-50 cell line, which iscapable of stable expression of rep/cap from an endogenous AAV promoter,is sequentially infected with a helper virus, preferably anE1-expressing, E2b defective adenovirus, to activate rep/cap and providehelper function, and an Ad-AAV hybrid virus, which efficiently transfersand replicates the AAV vector sequence. Preferably, the helperadenovirus expresses the adenovirus E1 gene product. The helper-infectedhost cell is then infected with a recombinant AdAAV hybrid virus, whichcomprises (1) adenovirus cis-elements necessary for replication andvirion encapsidation; (2) AAV sequences comprising the 5′ and 3′ ITRs ofan AAV, these AAV sequences flanked by the adenovirus sequences of (1);and (3) a selected gene operatively linked to regulatory sequencesdirecting its expression, said gene and regulatory sequences flanked bythe AAV sequences of (2). Preferably the sequence and timing of AAVrep/cap induction by the action of the helper virus relative to vectorreplication is balanced by infecting the cell line with the helper virusprior to infection with the hybrid virus. In one scaled-up embodiment,the infections are performed in bioreactors containing B50 cells adaptedfor growth in suspension. Following infection with a helper virus andtransfection with the hybrid virus or vector, the host cell is thencultured under standard conditions, such as described in e.g., F. L.Graham and L. Prevec, Methods Mol. Biol., 7:109-128 (1991). Desirably,once the rAAV is identified by conventional means, it may be recoveredand purified.

[0052] Important aspects of rAAV production for in vivo applicationsaccording to this method are purification and characterization of theproduct. Potential contaminants include rcAAV, cellular DNA, thehelper(s) and/or hybrid virus. The methods of this invention avoid themost troublesome contaminate, i.e., rcAAV. Contaminating cellular DNA isminimized by pretreatment of the vector with DNase. Elimination ofadenovirus is preferably accomplished by a combination of steps. Heatdenaturation with sedimentation through cesium effectively eliminatesfunctional adenovirus (<1 pfu Ad/10¹¹ rAAV genomes) and substantiallydiminishes contaminating adenovirus genomes (<0.00004% of total DNA)when used in the method of this invention. Methods more amenable tolarge scale production, such as column chromatography, are also useful.

[0053] As described in the examples below, the biological potency of AAVprepared by this method was evaluated in mice using erythropoietin (Epo)as an easily detected and quantified secreted protein, andbeta-galactosidase (lacZ), as an easily detected histochemical marker.Recombinant AAV produced by an embodiment of a method of this inventionperformed better (i.e., were more infectious) than rAAV made byconventional procedures of 293/cotransfection, when tested in mice.Furthermore, transgene expression from the rAAV produced by the presentmethods increased in proportion to vector dose. See, e.g., Example 5below.

[0054] The following examples illustrate several preferred methods ofthe invention. While the examples below employ the B50 cell line and awildtype adenovirus as helper, those of ordinary skill in the art willunderstand that any cell having the elements described above may besubstituted for the B50 cell line, and any helper fulfilling theabove-described helper functions (i.e., activating rep/cap expression)may be substituted for the wildtype adenovirus. These examples are thusillustrative only and are not intended to limit the scope of theinvention.

EXAMPLE 1 CONSTRUCTION AND CHARACTERIZATION OF THE B-50 REP/CAPCOMPLEMENTING CELL LINE

[0055] The inventors stably transfected into a number of cell lines,rep/cap containing plasmids in which rep and cap are expressed from aninducible promoter. These included the endogenous P5 promoter of AAVinduced by E1a of adenovirus as well as the heterologous promoters fromthe mouse metallothienien gene induced by divalent cations, and the longterminal repeat of murine mammary tumor virus induced byglucocorticoids. To overcome the problems associated with conventionalrAAV production methods involving transfection of cis and trans plasmidsin combination with helper adenovirus infection, three plasmid vectorswere constructed for generating rep/cap cell lines.

[0056] Each plasmid vector contained a neomycin selective marker geneand expressed the AAV rep/cap genes driven by either their native P5promoter (pP5-Rep/Cap), or the zinc-inducible sheep metallothioninepromoter (pMTRep/Cap), or the dexamethasone (Dex)-inducible mousemammary tumor virus (MMTV) promoter (pMMTV-Rep/Cap). These rep/capexpressing plasmids were derived from previously published constructsused to create an Ad E4-orf6 cell line in which the P5 rep/cap fragmentof pSub201 was replaced with orf6. Specifically, for construction ofpMT-Rep/Cap, ORF6 sequence was removed from pMTE4ORF6 plasmid [G. P. Gaoet al, J. Virol., 70:8934-8943 (1996)] by BamHI digestion and replacedwith a 4.1 kb rep/cap fragment which was prepared by PCR amplificationusing the pSub201 plasmid [Samulski, R. J. et al., J. Virol.,63:3822-3828 (1989)] as a template.

[0057] pMMTV-Rep/Cap was constructed in the same way as pMT-Rep/Cap,except that the pMMTVE4ORF6 plasmid was used as the vector backbone. Forconstruction of P5-Rep/Cap, the MT promoter and ORF6 sequences wereremoved from the pMTE4ORF6 plasmid by EcoRI/BamHI digestion and replacedwith a 4.3 kb P5-Rep/Cap fragment which was isolated from the pSub201plasmid by XbaI digestion. Plasmid construction involved conventionalgenetic engineering methods, such as those described in Sambrook et al,cited above.

[0058] First, the functionalities of these constructs were confirmed bytransient transfection into 10-3-rAAVLacZ cells, a 293-based cell linecontaining integrated forms of AdE4-ORF6 and rAAVLacZ genes [James M.Wilson's laboratory, The University of Pennsylvania] in the presence orabsence of wildtype adenovirus type 5 and appropriate inducers, i.e.,150 μM ZnSO₄ for pMT-Rep/Cap, and 10 μM dexamethasone for pMMTV-Rep/Cap.Transfection of all three constructs into 10-3-rAAVLacZ cells led torescue of rAAVLacZ from the cells. This demonstrated that all threeconstructs produced functional rep/cap proteins.

[0059] Second, all three constructs were stably transfected into bothHeLa and A549 cells, which are available commercially from the AmericanType Culture Collection. Stable transfectant colonies were selected inthe presence of culture media containing the antibiotic G418 (Geneticin)for two weeks, and were expanded individually.

[0060] These clones were evaluated for the capacity to transcomplementin the production of rAAVLacZ by transiently transfecting the cells withprAAVLacZ, a cis plasmid for producing rAAV LacZ which contains anCMV-β-galactosidase-SV40 poly A minigene cassette flanked by AAV ITRs[Fisher K. J. et al, J. Virol., 70:520-532 (1996)], via DOTAP in thepresence of Ad5Wt and appropriate inducers. For clones transfected withpMT-Rep/Cap, 150 μM ZnSO₄ was added to the culture medium 24 hours priorto adenovirus infection at MOI of 5. For clones transfected withpMMTV-Rep/Cap, 10 μM dexamethasone was added to the culture medium 24hours prior to adenovirus infection at an MOI of 5. Infection with Ad5induced expression from the P5 promoter. Seventy-two hourspost-transfection/infection, the infected clones were harvested whenfull CPE was demonstrated. The viral lysate of each clone was made bythree rounds of freezing and thawing. One-tenth of each viral lysate washeat-incubated at 56° C. for 30 minutes and used to infect 84-31 cells,an E1/E4-double complementing cell line [Gao et al, cited above].Twenty-four hours post infection, the cells were stained with5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) forβ-galactosidase expression [see e.g., J. Price et al, Proc. Natl. Acad.Sci, USA 84:156-168 (1987)] and the percentage of blue cells was scored.Clones scoring as positive were subject to a number of additionalstudies including DNA hybridization, immunocytochemistry, and Westernanalysis for E1a, rep, and cap after infection with wild type Ad5.

[0061] The results are summarized as follows: 708 total G418^(R)colonies were picked. From these, 618 clones were subcultured. 515 ofthe 618 clones survived expansion and screening. From the screen, only 8positive clones were able to transcomplement rep/cap. One of these eightclones, designated B-50 (created by transfecting the P5-rep/cap plasmidinto HeLa cells) was found to express rep and cap gene products at highlevels. These 8 clones were further characterized for their geneticconstitution, rep/cap protein expression upon induction, and rAAVproduction.

[0062] B-50 was the only clone in which the rep/cap gene sequence wasdetected by Southern blotting of genomic DNAs from the cell linesdigested with BamHI. The copy numbers of replcap genes and the forms ofthe genes (episome v.s. integrated) in the cell were confirmed byrepeating the Southern Blot with both total genomic and Hirt's DNAprepared from B-50 cells. DNA hybridization analysis revealed fivecopies of the plasmid arranged in a head-to-tail concatamer (data notshown). In B-50 cells, the replcap genes are stably integrated into thegenome and there is no episomal form of rep/cap sequences detectable bySouthern blot.

[0063] In addition, high levels of rep/cap protein expression wasdetected in B-50 by Western blotting (i.e., yielding 100-fold morevector than the others). Double immunofluorescent staining of adenovirusinfected B-50 cells for the AdE1b early protein or late proteins andrep/cap proteins demonstrated that B-50 is a homogenous cell line. Thestability of the B-50 cell line was demonstrated by its rAAVproductivity, comparing passage 5 to passage 15.

EXAMPLE 2 THE KINETICS OF REP/CAP INDUCTION IN B50 CELLS

[0064] The kinetics of rep/cap induction in B50 cells was determinedfollowing infection with adenovirus. B50 cells were infected with wildtype Ad5 at an MOI of 10 and total cellular proteins were prepared atdifferent time points post-infection. Samples (50 μg) were fractionatedon 10% SDS-PAGE gels and electrotransferred into nitrocellularmembranes. Rep and cap proteins were detected with ECL system (AmershamLife Science) using mouse monoclonal antibodies clones 259.5 and B1(American Research Products, Inc.) respectively. Adenovirus E1 proteinwas detected by a mouse monoclonal antibody against Ad2 E1a (OncogeneScience). The expression of E1a, rep protein, and cap proteins wereobserved on the gel (data not shown) as a function of time afterinfection and are summarized below.

[0065] In the absence of adenovirus, rep/cap proteins are not expressed.Infection with adenovirus results in a temporally regulated program ofgene expression with E1a protein peaking at 20-24 hours, followed by theinduction of substantial quantities of all rep and cap proteins, whichreach maximal levels by 44 hours and are sustained until the cells reachfull cytopathology by 62 hours. Levels of rep/cap expression areequivalent to that observed following a wild type of AAV infection,although profiles are different in that proportionally greater rep 52/40are expressed relative to rep 78/68 and more VP3 is expressed relativeto VP 1 and VP2, as compared to the profile of a wildtype infection.

EXAMPLE 3 RECOMBINANT AAV PRODUCTION IN THE B-50 CELL LINE

[0066] The productivity of rAAV expression by B-50 was assessed byinfecting the cells at different MOIs with either (a) recombinant hybridAdAAV virus and wildtype adenovirus type 5 (Ad5Wt) simultaneously; or(b) Ad5Wt alone for 8, 12, 16, 20 and 24 hours prior to infection withthe hybrid AdAAV virus

[0067] A. Hybrid Virus Construction

[0068] The procedure for constructing hybrid AdAAV vectors has beendescribed [Fisher et al, Hum. Gene Ther. 7:2079-2087 (1996)]. Briefly,rAAV genome containing a minigene cassette with the gene of interest andflanking AAV ITRs was isolated by PvuII restriction endonucleasedigestion from prAAV cis plasmid and cloned into the EcoRV site of theshuttle plasmidpAdBg/II. Transgenes included green fluorescent protein(GFP), LacZ, and a cDNA encoding erythropoietin from rhesus monkeysisolated by RT-PCR of RNA from pituitary. Each vector expressed thetransgene from the immediate early promoter of cytomegalovirus. Theresulting constructs consist of the 5′ sequence of Ad (map unit 0-1), acopy of rAAV genome, and Ad sequence spanning map units 9-16.1.Adenoviral DNA was prepared for cotransfection by digestion with ClaI.The supercoiled plasmid DNA of pAdAAV was used for cotransfection usinga standard calcium phosphate precipitation protocol and recombinanthybrid viruses were isolated as described in Fisher, cited above.

[0069] As one example the hybrid virus, H5.010rAAVLacZ (alsoAd.AV.CMVLacZ) was created by cloning an rAAV genome in place of E1 inan Ad5 based virus. This hybrid virus is described in detail in Example1 of International publication WO96/13598, cited above and incorporatedby reference herein. It contains the 5′ AAV ITR (bp 1-173) from AAV type2, a CMV immediate early enhancer/promoter [Boshart et al, Cell,41:521-530 (1985), an SV40 intron, E. coli beta-galactosidase cDNA, anSV40 polyadenylation signal and 3′ AAV ITR from AAV2, flanked byadenovirus type 5 map units 0-1 on one side and substantially all ofm.u. 9-100 on the other.

[0070] B. Experimental Protocol

[0071] B50 cells were seeded in 60 mm² plates at a density of 2×10⁵cells per plate overnight. The cells were infected with wild type Ad5and hybrid Ad.AV.CMVLacZ simultaneously. Alternatively, the cells wereinfected with wild type Ad5 at 8, 12, 16, 20 and 24 hours prior toinfection with hybrid Ad.AV.CMVLacZ at an MOI of 10.

[0072] Total cellular lysates were prepared at 48 hours post hybridAd.AV.CMVLacZ infection by 3 rounds of freezing/thawing andheat-inactivation at 56° C. for 1 hour. Production of rAAVLacZ in eachsample was determined by infecting 84-31 cells, an E1/E4-doublecomplementing cell line permissive for rAAV transduction, with thecellular lysate in serial dilutions for 20 hours. Twenty-four hours postinfection, the cells were histochemically stained with X-gal and LacZForming Units (LFUs), i.e., blue cells, were counted.

[0073] The production of rAAV under different conditions is shown inFIG. 2A, with the rAAV represented as lacZ Forming Units per cell on Yaxis vs. interval between helper and hybrid infection. These experimentswere repeated on two occasions with identical results.

[0074] C. Experimental Protocol

[0075] In another experiment, the B50 cells were seeded as describedabove and infected with wild type Ad5 24 hours prior to Ad.AV.CMVLacZ atan MOI of 10. Total cellular lysates were prepared at 2 to 72 hours postthe hybrid virus infection by 3 rounds of freezing/thawing. Duplicatesof each sample were either treated at 56° C. for 1 hour or not treatedand assayed for LFU as above.

[0076] The results are reported in FIG. 2B. The dotted line representsgrowth kinetics of the hybrid which is defined as the transductionmeasured after heat inactivation. The solid line indicates excision andamplification of rAAV genomes and production of vector as measured bytransduction after heat inactivation.

[0077] D. Experimental Protocol

[0078] In another experiment, B-50 cells were infected at 0, 8, 12, 16,20, and 24 hours post Ad5wt infection (MOI of 20) with rAd.AV.CMVLacZ.Cell lysates were prepared at 24, 48 and 72 hours post infection by thehybrid AdAAV vector described in Example 2 for LFU assays on 84-31cells.

[0079] The data was reported in FIG. 1, and shows that where B-50 cellswere pre-infected at an MOI of 20 with Ad5wt for 24 hours, and theninfected with rAdAAV for 48 hours, B-50 cells can produce at least 10LFU of rAAVLacZ per cell.

[0080] E. Experimental Protocol

[0081] In still another experiment, B-50 cells in 60 mm plates (about1.5×10⁶ cells per plate) were infected with Ad5wt at MOI of 1, 2, 5, and10 for 24 hours and then superinfected with H5.010rAAVLacZ at thecorresponding MOIs. The cells were harvested at 18, 24, 36, 48 and 60hours post H5.010rAAVLacZ infection and the lysates were used todetermine LFU on 84-31 cells. The results of such an LFU assay are shownbelow in Table 1. The figures reported in column 3 are the total LFUobtained from samples containing 1.5×10⁶cells. TABLE 1 Hours post hybridMOI infection Total LFU LFU/cell 1 18 1.6 × 10⁵ 0.1 1 24 2.0 × 10⁶ 1.4 136 1.3 × 10⁷ 8.8 1 48 1.8 × 10⁷ 12 1 60 1.7 × 10⁷ 11.5 2 18 8.0 × 10⁴0.05 2 24 9.8 × 10⁶ 6.5 2 36 1.9 × 10⁷ 12.3 2 48 3.1 × 10⁷ 21 2 60 2.0 ×10⁷ 13.5 5 18 1.4 × 10⁶ 1.0 5 24 2.1 × 10⁷ 14 5 36 4.7 × 10⁷ 32 5 48 3.5× 10⁷ 23 5 60 2.8 × 10⁷ 19 10 18 1.2 × 10⁶ 0.8 10 24 2.0 × 10⁷ 13 10 365.2 × 10⁷ 35 10 48 2.7 × 10⁷ 18 10 60 2.8 × 10⁷ 19

[0082] F. Experimental Protocol

[0083] The method of rAAV production of the present invention wasscaled-up to ˜10⁹ cells (100×15 cm² plates) using B-50 as the cell lineand Ad5wt as the helper. This method was compared to the performance ofthe standard method of rAAV production which is based on transientcotransfection of vector and rep/cap plasmid into 293 cells togetherwith adenovirus infection (called 293/cotransfection method) [See,Fisher (1996) cited above]. This large scale production of AAVexpressing green fluorescent protein (GFP) or rhesus monkeyerythropoietin (rhEpo) was also attempted with the method of thisinvention, using B-50 as the cell line and the E2b defective adenovirussub100r [provided by Jerome Schaack, UCHSC; Schaack, J. et al, J. Virol.69:4079-4085 (1995)]] as the helper. These results were compared withthe standard 293/cotransfection method using the same hybrid AAVplasmids and a replication defective beta galactosidase expressingrecombinant adenovirus (ΔE1lacZ) as helper.

[0084] Table 2 summarizes the results of rAAV expressing GFP (AAVCMVGFP)under the control of the CMV promoter and rAAV expressing rhEpo underthe control of the CMV promoter (AAVCMVrhEpo). Purified preparationswere analyzed for rAAV genomes and transduction. Table 2 reports acomparison of rAAV production, in which the total yield of rAAV ispresented based on the production of 10⁹ cell (i.e., 100×15 cm² plates).Total transduction units (i.e., the number of infected cells) in column3 are reported as determined by in situ analysis of reporter geneexpression following limiting dilution. Total genome copies in column 4(i.e., number of virus particles/10⁹ cells) are determined by DNAhybridization. The abbreviation Tu/GC in column 5 representstransduction units/genome copies of average values. TABLE 2 Total TotalTransductio Genome No. of rAAV Helper n Units Copies Tu/GC Preps. Methodof the Invention AAVCMVGFP Ad5Wt 1.9 × 10¹⁰ 2.2 × 10¹³ 685 2 5.2 × 10₁₀2.5 × 10¹³ AAVCMVGFP Sub100r 7.8 ± 0.8 × 10¹⁰ 5.9 ± 0.6 × 10¹⁴ 7564 3AAVCMVrhEpo Sub100r N/A 7 × 10¹³ N/A 1 3.4 × 10¹³ Standard293/cotransfection method AAVCMVGFP ΔE1lacZ 3.3 ± 3.4 × 10⁸ 6.4 ± 0.4 ×10¹² 19393 4 AAVCMVrhEpo ΔE1lacZ N/A 9.3 ± 7.4 × 10¹⁴ N/A 3

[0085] The standard method yielded 3.3×10⁸ transducing units and6.4×10¹² genome copies per 10⁹ cells for the rAAV containing GFP, whichis consistent with published results [Fisher (1996), cited above]. Themethod of the present invention yielded 100-fold more rAAV containingGFP, based on the transduction titers, with a 28-fold improvement inpotency or number of cells transduced with infectious rAAV, as measuredby a reduction in the ratio of genome copies to transduction units.Yields of GFP vector were increased with sub100r over what was obtainedwith wild type Ad (transducing units increased 2-fold and genome copiesincreased 25-fold), although potency, as measured by the ratio of vectorgenomes to transduction units, was decreased 11-fold. The total yield ofAAV particle numbers and number of infectious units was increasedoverall.

[0086] A 6-fold increase in genome copies was obtained with an AAVvector expressing rhesus monkey Epo when comparing the production methodof the present invention using the sub100r Ad helper to the standard293/transfection method. In general, the production method of thepresent invention has demonstrated 20- to 100-fold increases in genometiter (i.e., genome copies per ml) over that obtained with the standard293/cotransfection method for other AdAAV hybrid vectors tested, whichvectors contained the same AAVCMV backbone, but different transgenes,such as growth hormone and ornithine transcarbamylase, among others(data not shown).

[0087] A number of replication defective, E1 expressing adenoviruseswere evaluated as potential substitutes for the wild type Ad5 helper inthe production of AAV-GFP from the hybrid vector (data not shown). Asexpected, production of rAAV was substantially diminished with helperviruses defective in E2a or E4, both of which are necessary for AAVreplication [Kotin, R. M. Hum. Gene Thera. 5, 793-801 (1994)]. However,temperature sensitive mutations in the E2b gene [i.e., ts 149 [Myers, M.W. et a, J. Virol. 35, 65-75 (1980)] and sub100r did not diminish theyield of rAAV. As reported in the prior art, those adenovirus genesnecessary for maximal AAV production levels appear to include E1, E2a,E4 and VAI.

[0088] G. Experimental Protocol

[0089] Additional experiments relating to the production method of thepresent invention demonstrated high levels of rAAV, which exceed thoseobtained using the standard 293/cotransfection method with the wild typeAAV as helper in control experiments. Such high yields as describedabove span from over 2 fold to over 20 fold the yields of the controls(e.g., a genome copy of greater than 1×10³ virus particles per cellthrough greater than 1×10⁶ particles/cell, see above). Cells of thisinvention, i.e., B-50 cells were infected with the lacZ hybrid virus(Ad.AV.CMV LacZ) 24 hours after wild type Ad helper and lysates weresubsequently harvested and analyzed for production of lacZ-containingAAV versus amplification of lacZ Ad.AAV hybrid virus. Specificmeasurements included lacZ transduction before heat denaturation (whichincludes hybrid virus and rAAV) and after heat denaturation (whichrepresents rAAV). Contribution of the hybrid virus to transduction isthe difference between the two transduction titers (i.e., before andafter heat denaturation).

[0090] Representative kinetics of these experiments are shown in FIG. 2Bwhich indicate that the hybrid undergoes an exponential 100-foldamplification between about 12 and about 24 hours after seeding, whichimmediately precedes a second round of AAV amplification of equalproportion. The hybrid virus is amplified several orders of magnitude inB50 cells prior to or concurrent with rescue and replication of the rAAVgenome.

[0091] H. Summary of Experimental Results

[0092] The introduction of a helper which can transactivate (e.g.,activate the promoter controlling expression of rep/cap in the cell) therep/cap expression in the stable rep/cap expressing cell line of thisinvention, e.g., B-50 cells, and provide helper functions for AAVreplication, followed by the introduction of the Ad-AAV hybrid virusprovides the components necessary for rAAV production. Indeed, infectionof B50 with a hybrid virus that contains an AAV vector expressing GFPbut without provision of the transactivating adenovirus E1 gene, did notyield detectable rAAV. Further, as demonstrated, the timing of theprovision of the transactivating agent, which can be an adenovirus E1gene (by use of E1-expressing wildtype or modified adenoviruses, or byproviding the Ad E1 gene in another virus vector) or which can be byprovision of other virus transactivators (e.g., herpes, CMV, vaccinia),prior to introduction of the AdAAV hybrid vector can influence the yieldof rAAV.

[0093] When B-50 cells were infected with Ad5Wt and H5.010 rAAVLacZ atthe same time, there was no rAAVLacZ produced, presumably because thereplication of H5.010rAAVLacZ in the presence of Ad5Wt overrides theproduction of rAAVLacZ. The rAAV genome can be rescued fromH5.010rAAVLacZ-infected B-50 cells only when the cells were infectedwith Ad5Wt prior to H5.010rAAV LacZ infection. When the B-50 cells wereinfected with Ad5Wt first, onset of rep/cap protein expression inhibitedadenoviral vector replication and promoted rAAVLacZ production, whichforced the balance towards rAAV production instead of H5.010rAAVLacZreplication. Rep protein expression continues until full cytopathiceffect (CPE), while cap protein expression becomes detectable at 20hours post-infection, reaches the peak at 40 hours post-infection, andthen reaches a plateau. The yield of rAAVLacZ under these conditions isbetween about 1×10³ to 5×10³ virus particles per B-50 cell (i.e., 1-5LFU).

[0094] Thus, this analysis of one embodiment of a method of theinvention shows that rAAV productivity in B-50 cells infected with Ad5wtand AdAAV hybrid virus demonstrated that Ad5wt infection of B-50 cellsprior to rAdAAV infection produces a high yield (i.e., greater than1×10³ virus particles to greater than 1×10⁶ virus particles) ofinfectious rAAV. Such yields are between greater than 2-fold to greaterthan 20 fold the reported yields of the prior art methods.

[0095] The temporal relationship between rep/cap induction and AAVrescue and replication as well as infectious dose, i.e., themultiplicity of infection (MOI) of the helper and AdAAV hybrid viruses,can be readily adjusted to optimize rAAV production, depending on thecell line, helper and AdAAV hybrid used. Such adjustments areaccomplished by performing experiments varying the times of infectionand MOIs, such as those described above and reported in FIGS. 2A and 2B.In the embodiments described in FIG. 2A, for example, these experimentsvarying the time between infection of the wild type and hybrid virusesdemonstrated a substantial increase in rAAV production with maximalyields obtained, when B50 cells were infected with hybrid virus 24 hoursafter the helper (see, e.g., FIG. 2A).

[0096] The method of this invention utilizing endogenous AAV promotersin the host cell line in combination with an E1 expressing, helperadenovirus simulates the biology of a wild type AAV infection.Presently, desirable conditions for rAAV production using the B-50 cellline, wildtype adenovirus and the Ad.AAVCMVLacZ hybrid virus as thecis-acting vector include the following:

[0097] (a) infect B-50 cells with Ad5wt at a MOI of at least 5. An MOIof 10 is also desirable, although the less wildtype adenovirus employedin the method, the easier is the purification of rAAV from helper) forbetween 12 to 24 hours. As shown above for this embodiment, 24 hoursappears to be most desirable to enhance rAAV yield;

[0098] (b) infect the cells with the hybrid Ad.AAVCMVLacZ virus at anMOI of at least 5 for an additional 36 hours. One of skill in the artgiven the teachings of this invention can easily adjust these dosage andinfection timing parameters to provide optimal conditions for otherembodiments of this invention employing other cell lines than B-50,other hybrid viruses and other helpers. The performance of the tests todetermine optimal production conditions do not involve any degree ofundue experimentation.

EXAMPLE 4 RECOMBINANT AAV PRODUCED BY THE HYBRID VIRUS IS FREE OFREPLICATION COMPETENT AAV

[0099] The traditional method for producing rAAV frequently yieldsreplication competent AAV (rcAAV) through nonhomologous recombinationthat occurs during the transient transfection. This example demonstratesthat formation of rcAAV is decreased by using the production method ofthis invention, because the transfected rep/cap genes in the chromosomalDNA of the cell line of the invention are sequestered from theadenovirus encoded AAV vector DNA of the hybrid virus.

[0100] A. Production and Purification of rAAV.

[0101] rAAV was generated by plasmid transfection of the cis plasmid(i.e., which carries the AAV cassette) and trans plasmid (i.e., whichcarries the rep/cap genes) in 293 cells infected with E1 deletedadenovirus and isolated following heat denaturation and CsCl gradientpurification, as described by Fisher et al, Nature Med. 3:306-312(1996). Alternatively, rAAV was produced using the B50 cell line andAdAAV hybrid virus according to the method of this invention. B50 cellsseeded in 150 mm² plates at a density of 1×10⁷ cells per plate wereinfected with either wild type Ad5 or sub100r virus [Schaack, citedabove; i.e., a temperature sensitive mutation in E2b30] at an MOI of 10to 24 hours, and then with the AdAAV hybrid vector at the same MOI foran additional 48 hours. The cells were harvested for rAAV preparationand CsCl gradient purification as described in Fisher, cited above.

[0102] Following the heat denaturation and CsCl centrifugation, the rAAVwere subjected to several analyses. The total amount of rAAV genomes wasquantitated by DNA hybridization as described by Fisher, cited above.Transducing titer was determined by exposing the AAV permissive cellline 84-31 to limiting dilutions of rAAV and analyzing the monolayer forfoci of transgene expressing cells twenty-four hours later.Contaminating adenovirus was assessed by a plaque forming assay at asensitivity of 1 adenovirus/10¹¹ rAAV genomes. No contaminating Ad wasdetected at these levels.

[0103] B. Assay for contaminating rcAAV

[0104] The assay for replication competent AAV (rcAAV) is based onamplification of 293 cells in the presence of adenovirus through twopassages followed by analysis of the resulting lysates for rep DNA byspecific hybridization. Briefly described, the rAAV prep (10¹⁰-10¹¹genomes) is mixed with various quantities of wild type AAV (0 to 10⁴genomes) in the presence or absence of wild type adenovirus at an MOI of5 which was incubated with 5×10⁶ 293 cells in a 100 mm² plate. The cellswere harvested and total DNA was isolated for hybridization or theclarified crude lysates were prepared for a second round ofamplification. After heat-inactivation of adenovirus at 56° C. for 1hour, one-tenth of the crude lysate was inoculated in a fresh plate of293 cells in the presence or absence of adenovirus for the second roundof amplification. The cells were harvested 72 hours later and total DNAwas prepared as the first amplification. Total DNA (10 μg) from thefirst and second amplifications was analyzed for rep sequences by DNAhybridization following digestion with HindIII endonuclease. The blotwas hybridized to a ³²P-labeled 2.7 kB HindIII/XbaI fragment of pAd/AAVcontaining cap sequence.

[0105] A sensitive contamination assay was developed to detect low levelrcAAV in the presence of adenovirus to amplify the rcAAV. 293 cells in100 mm² plates were infected with 10¹, 10², 10³, and 10⁴ genome copiesof wild type AAV2 in the presence and absence of wild type Ad5 at an MOIof 5 for 72 hours. Assays were also performed in the presence of 293cell lysate to evaluate possible interference in the assay. Total DNAswere prepared from two-thirds of the infected cells. The other one-thirdof cells were lysed in the infection medium by three rounds offreezing/thawing and one-fifth of each lysate was heat-inactivated forthe second amplification in 293 cells for another 72 hours. After thissecond passage, total DNAs were extracted again. The lysate is evaluatedfor rep DNA by Southern blot hybridization analysis as follows: totalDNA (10 μg) digested with HindIII was fractionated, blotted and probedwith a 2.7 kb Cap specific fragment. Analysis of DNA following the firstand second amplification was shown in the resulting autoradiograms (notshown). Digestion of the trans plasmid with XbaI/HindIII releases afragment that comigrates with the amplified and restricted rcAAV. Thisassay detects as little as 10 wild type AAV genomes. Similar levels ofrcAAV contamination were observed in transfection experiments in whichthe trans plasmid was modified to minimize recombination or when theadenovirus helper was replaced with an Ad plasmid (data not shown).

[0106] Detection of rcAAV in large scale production lots was made asfollows: 2×10¹⁰, 2×10⁹, and 2×10⁸ genome copies each of two purifiedrAAV stocks were used for 1 round of amplification in 293 cells.rAAvlacZ was produced by 293/cotransfection, whereas rAAVGFP wasproduced by B50 hybrid system. The analysis of the first round amplifiedlysate was made in an autoradiogram (not shown). Analysis of rAAVproduced by 293/cotransfection methodology consistently demonstratedrcAAV at levels equivalent to 1/10³-10⁶ of the total rAAV preparation;no rcAAV (<1/10⁹) was detected from rAAV preparations made by the methodof this invention employing the B50 cell line and hybrid AdAAV. No rcAAVwas seen following another round of amplification.

[0107] Sensitivity of the contamination assay in the presence of vectoras assessed by spiking stocks of rAAV produced by the method of thisinvention using B-50 and with limiting quantities of wild type AAV. Todetermine the potential interference of rAAV genomes on the detection ofrcAAV, 1, 10¹, 10², 10³, and 10⁴ genomes of wild type AAV were spikedinto either 2×10¹⁰ or 2×10¹¹ genomes each of rAAVGFP produced byB50/shuttle virus system for 2 rounds of amplification. Total DNA wasextracted from the infected cells (10 μg) of the second roundsamplifications and digested with HindIII prior to Southern blotanalysis. As little as 10 wild type AAV genomes can be detected in2×10¹⁰ rAAV genomes, although the sensitivity of the assay for detectionof contaminating rcAAV is reduced in the presence of 10-fold higherAdAAV vector. The final preparations contained less than 1 transducingadenoviral particle per 10¹¹ AAV genomes.

EXAMPLE 5 IN VIVO USE OF rAAV PRODUCED BY THE HYBRID VIRUS

[0108] A comparative study of rAAV preparations was performed byintroducing rAAV expressing either rhesus monkey Epo or lacZ intoskeletal muscle of mice. In this experiment, intramuscular injection ofAAV vector encoding erythropoietin into skeletal muscle of mice resultedin supraphysiologic levels of hormone in serum that was stained andcaused polycythemia.

[0109] More particularly, recombinant AAV expressing rhesus monkey Epoor lacZ from a CMV promoter was analyzed in either immune deficient mice(i.e., Rag1^(−/−)) or immune competent mice (C57BL/6) as follows.C57BL/6 or Rag^(−/−)mice (4/group) were injected with 1×10₁₁ genomecopies/50 μl of rAAV-Epo into the tibialis anterior muscle. The rAAV-Epowas produced according to the standard 293/cotransfection method oraccording to the method of this invention employing the B50 cell lineand the AdAAV hybrid.

[0110] Serum from the rAAV-Epo injected animals was harvested andanalyzed for virus derived Epo using commercially available ELISA(Quantikine IVD, R & D Systems) that cross-reacts with rhesus monkeyEpo. Recombinant human erythropoietin served as a standard. Hematocritswere determined using microcapillary tables followed by centrifugationin an IEC Micro-MB centrifuge. Equivalent doses (1×10¹¹ genomes) of eachrAAV-Epo preparation yielded similar elevations in hematocrit, althoughthe B50/hybrid rAAV-Epo vector yielded 4-fold more serum Epo than therAAV-Epo vector derived from 293/co-transfection. See, e.g., FIGS. 3Aand 3B. The B50/hybrid produced rAAV-Epo was more infectious that therAAV-Epo produced by the conventional method.

[0111] Mice received various preparations of rAAV-lacZ by directinjection into the tibialis anterior, i.e., 293/co-transfection(1.7×10¹⁰ genome copies) and B50/hybrid (1.7×10¹⁰ genome copies or3.8×10¹⁰ genome copies or 1.7×10¹¹ genome copies). Skeletal muscle washarvested five weeks later and evaluated for β-galactosidase expressionusing X-gal histochemistry. Identical levels of X-gal staining in thetibialis anterior were seen 4 weeks after immunocompetent mice wereinjected with 1.7×10¹⁰ genomes of vector derived from the B50/hybrid and293/co-transfection methods. Increasing the dose of the lacZ vectorproduced by the B50/hybrid method yielded a proportional increase inP-galactosidase staining without evidence of inflammation (data notshown). For example, infection with 1.7×10¹⁰ genome copies yielded froma visual inspection of the gel, about 5% stained muscle fiber. Incomparison, infection with 1.7×10¹¹ genome copies yielded from a visualinspection of the gel, about 50% stained muscle fiber. Similarly a3-fold increase in genome copies from the 5% baseline, yielded about 15%stained muscle fiber.

[0112] All documents cited above are incorporated by reference herein.Numerous modifications and variations of the present invention areincluded in the above-identified specification and are expected to beobvious to one of skill in the art. Such modifications and alterationsto the processes of the present invention are believed to be encompassedin the scope of the claims appended hereto.

What is claimed is:
 1. A cell useful for production of recombinantadeno-associated virus (AAV), wherein said cell has stably integrated inits chromosome at least two copies of an AAV rep gene and AAV cap geneunder the control of regulatory sequences comprising AAV P5 which islocated in the native AAV position upstream of the AAV rep gene andwhich directs expression upon activation with an exogenously introducedhelper.
 2. The cell according to claim 1, wherein the rep and cap genesare operatively linked to separate regulatory sequences.
 3. The cellaccording to claim 1, wherein the rep and cap genes are operativelylinked to the same regulatory sequences.
 4. The cell according to claim1, wherein the rep and cap genes are derived from different serotypes ofAAV.
 5. The cell according to claim 1, wherein the AAV P5 promoter isderived from a different AAV serotype than the rep and cap genes.
 6. Thecell according to claim 1, wherein said cell has at least three copiesof an AAV rep gene and an AAV cap gene stably integrated in itschromosome.
 7. The cell according to claim 1, wherein said cell has atleast four copies of an AAV rep gene and an AAV cap gene stablyintegrated in its chromosome.
 8. The cell according to claim 1, whereinthe helper is a member selected from the group consisting of anadenovirus, a herpes virus, a recombinant adenovirus, a recombinantherpes virus, an adenovirus gene, a herpes virus gene, an adenovirusgene product, and a herpes virus gene product.
 9. The cell according toclaim 1, wherein said at least two copies are in a head-to-tailconcatamer form.
 10. The cell according to claim 1, wherein said atleast two copies are in a head-to-head concatamer form.
 11. The cellaccording to claim 1, wherein the cell contains multiple copies of theregulatory sequences which control expression of the rep and cap genes.12. A method for producing recombinant AAV, said method comprising thesteps of (a) providing a cell useful for production of recombinantadeno-associated virus (AAV), wherein said cell has stably integrated inits chromosome at least two copies of an AAV rep gene and AAV cap geneunder the control of regulatory sequences comprising AAV P5 which islocated in the native AAV position upstream of the AAV rep gene andwhich directs expression upon activation with an exogenously introducedhelper, (b) introducing to the helper-containing host cell of (a) arecombinant hybrid virus, the recombinant hybrid virus comprising: (1) aselected transgene operatively linked to regulatory sequencescontrolling the transgene's expression, (2) AAV sequences comprising the5′ and 3′ ITRs of an AAV, wherein the 5′ ITR flanks one side of thetransgene and regulatory sequences of (1), and the 3′ ITR flanks theother side, and (3) an adenovirus cis-element selected from the groupconsisting of cis elements required for replication of adenovirusvirions and cis elements required for encapsidation of adenovirusvirions, said cis-elements flanking each AAV sequence of (2); wherebyrecombinant AAV is produced by the cell.
 13. The method according toclaim 12, wherein step (b) is performed about 24 hours after the helperis introduced to the host cell.
 14. The method according to claim 12,comprising the additional step of: (c) isolating from the hybridvirus-infected, helper-containing host cell a recombinant AAV comprisingthe transgene.
 15. The method according to claim 14, wherein step (c) isperformed 36 hours after the helper-containing host cell is infectedwith the hybrid virus.
 16. The method according to claim 12, wherein therecombinant AAV is produced at levels exceeding 1×10³ genome copies percell.
 17. The method according to claim 14, wherein the recombinant AAVis isolated in step (c) at a level exceeding 1×10⁴ genome copies percell.
 18. The method according to claim 12, wherein the recombinant AAVproduced is essentially homogenous and is essentially free ofreplication-competent AAV.
 19. The method according to claim 12, whereinthe host cell is a B-50 cell having ATCC Accession No. CRL
 12401. 20.The method according to claim 12, wherein the helper is a memberselected from the group consisting of an adenovirus, a herpes virus, arecombinant adenovirus, a recombinant herpes virus, an adenovirus gene,a herpes virus gene, an adenovirus gene product, and a herpes virus geneproduct.
 21. The method according to claim 20, wherein the helper is arecombinant adenovirus comprising an adenovirus E1 gene.
 22. The methodaccording to claim 21, wherein the recombinant adenovirus isreplication-deficient.
 23. The method according to claim 20, wherein thehelper comprises an adenovirus E1 gene.
 24. The method according toclaim 20, wherein the recombinant herpes virus is replication-deficient.25. The method according to claim 20, wherein the helper comprises is arecombinant virus selected from the group consisting of Herpes SimplexType I, Herpes Simplex Type II, Cytomegalovirus and Vaccinia.